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Application of GFP Imaging in Cancer Robert M Hoffman Laboratory Investigation (2015) 95, 432–452 & 2015 USCAP, Inc All rights reserved 0023-6837/15 $32.00 PATHOBIOLOGY IN FOCUS Application of GFP imaging in cancer Robert M Hoffman Multicolored proteins have allowed the color-coding of cancer cells growing in vivo and enabled the distinction of host from tumor with single-cell resolution. Non-invasive imaging with fluorescent proteins enabled the dynamics of metastatic cancer to be followed in real time in individual animals. Non-invasive imaging of cancer cells expressing fluorescent proteins has allowed the real-time determination of efficacy of candidate antitumor and antimetastatic agents in mouse models. The use of fluorescent proteins to differentially label cancer cells in the nucleus and cytoplasm can visualize the nuclear–cytoplasmic dynamics of cancer cells in vivo including: mitosis, apoptosis, cell-cycle position, and differential behavior of nucleus and cytoplasm that occurs during cancer-cell deformation and extravasation. Recent applications of the technology described here include linking fluorescent proteins with cell-cycle-specific proteins such that the cells change color from red to green as they transit from G1 to S phases. With the macro- and micro-imaging technologies described here, essentially any in vivo process can be imaged, giving rise to the new field of in vivo cell biology using fluorescent proteins. Laboratory Investigation (2015) 95, 432–452; doi:10.1038/labinvest.2014.154; published online 16 February 2015 ‘...during periods of revolution when the fundamental tenets cancer cells of a specific genotype or phenotype. For example, of a field are once more at issue, doubts are repeatedly the behavior of cancer stem cells labeled with GFP and non- expressed about the possibility of continued progress if one stem cells labeled with red fluorescent protein (RFP) can be or another of the opposed paradigms is adopted.’ simultaneously compared in vivo.3 The stroma and the cancer Thomas S. Kuhn in The Structure of Scientific Revolutions cells can be color-coded with fluorescent proteins. For (3rd Edition, The University of Chicago Press, Chicago and example, a transgenic mouse expressing GFP in all of its London, 1996). cells (or in specific cells such as endothelial cells) trans- planted with cancer cells expressing RFP enables the inter- INTRODUCTION action between the cancer cells and the host cells to be 4–6 When the Nobel Prize in chemistry was awarded for the visualized in real time. discovery of green fluorescent protein (GFP) in 2008, the The long wavelength excitation of some fluorescent pro- 7 Nobel Committee called GFP a guiding star for biochemistry teins, such as Katushka, also reduces the extent of photo- enabling processes that were previously invisible, such as bleaching and enables deep imaging. Therefore, real-time cancer cells spreading, to be strikingly visible. GFP and its red, imaging of tumor growth and metastasis can be carried out blue, yellow, and orange cousins have revolutionized bio- in the intact animal in deep tissues. For single-cell resolution, medical research and enabled every major disease, both cause reversible skin flaps as well as chronic-transparent window and effect, to ‘light-up’ in the laboratory for researchers to models can be used over many parts of the body (skin, brain, 8–10 visualize, understand, and eventually conquer them. Fluor- lung, liver, etc.). Real-time imaging with fluorescent proteins escent proteins allow us to visualize, in real time, all important is especially important when evaluating the efficacy of thera- 11 aspects of cancer in living animals, including the cancer cell cycle, peutics on metastasis, tumor recurrence, and cell cycle 12 mitosis, apoptosis, motility, invasion, metastasis, and angio- dependence. genesis. Multicolored proteins have allowed the color-coding of cancer cells growing in vivo and enabled the distinction of Properties of GFP and Other Fluorescent Proteins as host from tumor with single-cell resolution in vivo.1,2 Imaging Agents Fluorescent proteins of many different colors have now GFP and related fluorescent proteins are a family of proteins been characterized and these can be used to color-code with emission spectra ranging from 442 to over 650 nm.13 AntiCancer, Inc., Department of Surgery, University of California San Diego, San Diego, CA, USA Correspondence: RM Hoffman, PhD, AntiCancer, Inc., Department of Surgery, University of California San Diego, 7917 Ostrow Street, San Diego, CA 92111, USA. E-mail: [email protected] Received 14 August 2014; accepted 25 September 2014 432 Laboratory Investigation | Volume 95 April 2015 | www.laboratoryinvestigation.org PATHOBIOLOGY IN FOCUS GFP imaging and cancer RM Hoffman There are many spectrally distinct fluorescent proteins are being used. Spectral resolution enables imaging of tumor resulting in a palette of colors14 that can be simultaneously on deep tissues or high-resolution non-invasive visualization imaged. Fluorescent proteins range in size from 25 to 30 kDa of tumor blood vessels16 which express a fluorescent protein and form internal chromophores that do not require cofactors of a different color. or substrates to fluoresce. Fluorescent proteins generally have very high extinction coefficients ranging up to approximately Ex Vivo Imaging Using Fluorescent Proteins ¼ 95 000. In addition, fluorescent proteins have very high The first use of GFP for in vivo imaging, visualized cancer 15 quantum yields up to 0.8. These properties make cells growing in athymic nude mice in our laboratory.21 fluorescent proteins very bright. Two-photon absorption of Cancer cells were stably transfected with GFP and were trans- 10 GFP is important for deep-tissue imaging in vivo. Another planted into several mouse models, including orthotopic important feature of fluorescent proteins is the spectral models that have a high metastatic capacity. Metastases distinction between many members of the family, enabling of could be observed in any organ at the single-cell level in multicolor fluorescent proteins to be used simultaneously for unprocessed tissues. In addition, cells were visualized in the multifunctional in vivo imaging. Spectral separation imaging process of intravasation and extravasation. The visualization is also very useful to distinguish different colors including of single cancer cells in tissue is beyond the capabilities of 16 autofluorescence. standard histological techniques. In Vivo Imaging with Fluorescent Proteins Fluorescent proteins are so bright that simple equipment can Intravital Imaging Using Fluorescent Proteins be used for in vivo imaging. Macro-imaging studies require Before our laboratory’s pioneering use of GFP for in vivo equipment as simple as an LED flashlight with appropriate imaging, in vivo fluorescence imaging was limited to the vi- excitation emission filters.17 In vivo images can even be sualization of cells that were transiently labeled with vital acquired with a cell phone camera because fluorescent dyes. Use of fluorescent proteins, now allows the direct proteins are so bright! imaging of single cells in vivo for unlimited periods. A fluorescence light box with fiber-optic lighting at ap- Reversible skin flaps can be introduced onto different parts proximately 490 nm and appropriate filters, placed on top of of the animal to image single cancer cells or small colonies on 8 the light box (Figure 1), can be used to image tumors and internal organs. The skin flaps are reversed by simple metastasis that can be viewed with a camera to enable the suturing. External imaging can then be done through the images to be displayed on a monitor and digitally stored.18 relatively transparent body walls of the mouse, which include Excitation with a narrow band filter at approxi- the skull, at the single-cell level, by use of a simple mately 490 nm should be used. Fluorescence emission can fluorescence dissecting microscope or more sophisticated be observed through a 520 nm long-pass filter.18 equipment described in this review. Blood vessels growing on A powerful hand-held imaging device that inputs the image tumors can also be observed using skin-flaps because they 8 directly to a computer monitor can also be used.19 contrast with the fluorescence of the tumors. 22 A variable-magnification small animals imaging system With intravital microscopy, imaging through the opened (OV100, Olympus Corp., Tokyo, Japan), containing an MT- skin or body walls of live mice visualizes single cancer cells. All 20 light source (Olympus Biosystems, Planegg, Germany) steps in the metastatic process can be visualized. Individual, and DP70 CCD camera (Olympus), can be used for macro- nondividing cells as well as micro- and macrometastases can and subcellular imaging in live mice. The optics of the OV100 be clearly visualized and quantified. Cellular details, such as 23 fluorescence imaging system have been specially developed pseudopodial projections, can be clearly seen. In early 24 for macroimaging as well as microimaging with high light- studies, Farina et al observed cancer cell invasion at the gathering capacity. The objectives have high numerical single-cell level, including intravasation and extravasation, 25 aperture and long working distance. Individually optimized using GFP-expressing cells. Condeelis et al used confocal objective lenses, parcentered and parfocal, provide a 105-fold GFP imaging to observe the polarity of cancer cells magnification range for imaging of the entire body down to responding to chemotatic
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